Modified kisspeptin peptides and uses thereof

ABSTRACT

The present application provides synthetic modified peptides of five to seven natural or non-natural amino acids as well as pharmaceutical compositions comprising them, for use in the treatment a disease or disorder presenting behavioral abnormalities associated with impairment of sensory gating function, depression or cognitive impairment, particularly schizophrenia and Alzheimer&#39;s disease.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/384,685 filed on Sep. 11, 2014, which is a National Phase of PCTPatent Application No. PCT/IL2013/050248 having International filingdate of Mar. 14, 2013, which claims the benefit of priority under 35 USC§ 119(e) of U.S. Provisional Patent Application No. 61/610,698 filed onMar. 14, 2012. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 66606SequenceListing.txt, created on Jun. 8,2016, comprising 8,162 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to synthetic peptides and pharmaceuticallyacceptable salts thereof and pharmaceutical compositions comprisingthem, and their uses for treating diseases or disorders presentingbehavioral abnormalities such as Alzheimer's disease, or in particulardiseases or disorders associated with impairment of sensory gatingfunction such as schizophrenia, and/or for treating depression, and/orfor improving cognitive function.

BACKGROUND OF THE INVENTION

Schizophrenia is a severe mental disorder that affects about 1% of thepopulation. The etiology of schizophrenia is complex and involvesgenetic and environmental factors. Schizophrenia is often described interms of positive, negative and cognitive symptoms. Positive symptoms,such as delusions and hallucinations, are not normally experienced bymost individuals. Negative symptoms are deficits of normal emotionalresponses or of thought processes and cognitive symptoms are cognitivemalfunctions. Negative and cognitive symptoms respond less well tomedication than positive symptoms, and are contributing more to poorquality of life, functional disability, and the burden on others than dopositive symptoms.

Antipsychotics remain the current standard of care for mental disordersincluding schizophrenia and bipolar mania. The first generation ofantipsychotics (typical) such as haloperidol, inhibit dopamine D₂receptors and are moderately effective in treating positive symptoms ofschizophrenia, but may cause extrapyramidal movement disorders.Second-generation (atypical) antipsychotics inhibit D₂ receptors inconjunction with other receptors (notably 5-hydroxytryptamine 2A(5-HT_(2A)) receptors). Atypical antipsychotics have proved less likelyto cause movement disorders, but are associated with weight gain,prolactin and glucose elevation, and sedation. Recent data indicate thatagents that target metabolic glutamate receptors (mGluRs) couldrepresent a promising new class of antipsychotics, and such agents arenow being developed by several companies. Nevertheless, a large trialknown as CATIE, sponsored by the US National Institutes of Health, foundthat 74% of patients discontinue use within 18 months of therapy due toeither poor tolerability or incomplete efficacy, indicating a need fornovel therapies.

Kisspeptins (formerly known as Metastins), the products of the Kiss1gene, bind to a G protein-coupled receptor known as GPR54. Kiss1 wasoriginally identified as a human metastasis suppressor gene. Thekisspeptin-GPR54 signaling cascade was recognized several years ago ashaving a fundamental role in the regulation of sex hormones, and isconsidered to be the gatekeeper of puberty. Moreover, recent studiesindicated that kisspeptin serves additional physiological functions inthe cardiovascular system, angiogenesis, and in energy balance. In thehippocampus, kisspeptin enhances excitability of granule cells of thedentate gyrus and secretion of the growth factor brain-derivedneurotrophic factor (BDNF, Arai and Orwig, 2008). BDNF has importantfunctions in the development of the nervous system and in brainplasticity-related processes such as memory, learning, and drugaddiction. Various studies have shown possible links between BDNF andconditions such as depression, schizophrenia, obsessive-compulsivedisorder, Alzheimer's disease, Huntington's disease, Rett syndrome, anddementia, as well as anorexia nervosa and bulimia nervosa.

WO 2010/137022 of the same applicant discloses an elevation in mRNAexpression of Kiss1 during adolescence (8 weeks) in the hippocampus ofnaïve mice. Moreover, the regulation of Kiss1 expression in thehippocampus, unlike its expression in the hypothalamus, isimmune-dependent. Thus, functional relationships between the presence ofimmune deficiency (SCID mice), abnormal expression of kisspeptin in thehippocampus at puberty, and impaired processing of information was found(as measured by prepulse inhibition (PPI)) in these mice (Cardon et al.,2010). PPI is the phenomenon by which a low-intensity prepulse stimulusattenuates the response to a subsequent startle-eliciting noise.Deviations in PPI are commonly associated with the pathophysiology ofschizophrenia (Braff and Geyer, 1990) yet abnormal PPI was also found inother psychological disorders (Castellanos et al., 1996; Ornitz et al.,1992; Perry et al., 2007; Swerdlow et al., 1993; Swerdlow et al., 1995).

WO 2010/137022 also discloses that administration of the kisspeptinderived peptide Kp-10 (YNWNSFGLRF-NH₂) to SCID mice that have impairedPPI response and to several schizophrenia mouse models, improved PPIresponse levels. It is additionally disclosed that Kp-10 has a potentialanti-depressive activity by a tail suspension test and that Kp-10improves spatial learning and memory in a water maze in several mousemodels, indicating its contribution to broad aspects of cognitivefunctions such as learning and memory.

SUMMARY OF THE INVENTION

In accordance with the present invention, novel modified Kp-10 derivedpeptides were synthesized and found to improve PPI response in naïvemice and in schizophrenia and Alzheimer's mouse models.

The present invention thus provides a synthetic peptide selected from:

(i) a cyclic or linear peptide of the sequence:

(SEQ ID NOs: 2 and 1) R₁-Xaa₁-Gly-Xaa₂-Xaa₃-Xaa₄-Xaa₅-NHR₂ 

wherein:

-   -   Xaa₁ is selected from Phe, Ile, Leu, Val, Nle and analogs        thereof;    -   Xaa₂ is selected from Leu, Ile, Val, Nle, Phe and analogs        thereof;    -   Xaa₃ is selected from Arg, Lys, homo-Arg, homo-Lys, Orn and        analogs of Arg;    -   Xaa₄ is selected from Trp and analogs thereof;    -   Xaa₅ is selected from Tyr, His, O-methyl-Tyr and        2-hydroxy-3-methyl-Phe;    -   R₁ is selected from para-aminophenylalanine (Pap),        NH₂C₆H₄(CH₂)₁₋₃CO, Fmoc-Pap, FMS-Pap and C₅-C₂₀ acyl-Pap,        wherein said C₅-C₂₀ acyl is derived from a saturated or        unsaturated C₅-C₂₀ fatty acid linked to the α-amino group of        Pap; and    -   R₂ is H or (CH₂)₀₋₄CH₃, and        (ii) a linear peptide of the sequence:

(SEQ ID NO: 3) R₁-Xaa₁-Gly-Xaa₂-Xaa₃-Xaa₄-NHR₂ 

-   -   wherein:    -   Xaa₁ to Xaa₄ are as defined above;    -   R₁ is 9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-Fmoc (FMS);        and    -   R₂ is as defined above,    -   and pharmaceutically acceptable salts thereof.

The present invention further provides a cyclic synthetic peptide havingthe sequence:

The present invention further provides a linear synthetic peptide havingthe sequence: Fmoc-Phe-Gly-Leu-Arg-Trp-NH₂ (SEQ ID NO: 8).

The present invention also provides a pharmaceutical compositioncomprising the synthetic peptides of the invention or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier.

The synthetic peptides of the invention are also provided for use in thetreatment of a disease or disorder presenting behavioral abnormalitiesassociated with impairment of sensory gating function. The disease ordisorder can be schizophrenia.

The synthetic peptides of the invention are additionally provided foruse in the treatment of depression or of a cognitive impairment.

The synthetic peptides of the invention are also provided for use in thetreatment of Alzheimer's disease (AD).

The present invention also provides a method for the treatment of adisease or disorder presenting behavioral abnormalities associated withimpairment of sensory gating function, depression or cognitiveimpairment, comprising administering to a patient in need thereof aneffective amount of a peptide of the invention or a pharmaceuticallyacceptable salt thereof.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIGS. 1A-1E show the effect of synthetic peptides on prepulse inhibition(PPI). Male C57Bl/6J naïve mice were injected with synthetic peptides(light bars) or with 1% DMSO (control, black bars) in PBS; prepulseintensity was 69, 73, 78 or 81 decibels (dB). (A) 15 μg cyclic Peptide 2(SEQ ID NO: 10), n=12, control n=8, Repeated measure ANOVA, F (degreesof freedom) (1,18)=4.2, P=0.06. (B) 8.3 μg cyclic Peptide 5 (SEQ ID NO:12), n=8, control n=6; Repeated measure ANOVA, F (1, 12)=0.1, P=0.7. (C)8.75 μg cyclic Peptide 6 (SEQ ID NO: 6), n=8, control n=10; Repeatedmeasure ANOVA, F (1, 19)=4.7, P=0.047, * indicates P<0.05, Fisher LSDpost hoc analysis. (D) 15.3 μg Peptide 7 (SEQ ID NO: 13), n=12, controln=9, Repeated measure ANOVA, F (1, 18)=1.15, P=0.3. (E) 8.75 μg Peptide8 (SEQ ID NO: 8), n=17, control n=16, Repeated measure ANOVA, F (1,31)=5.3, P=0.028, * indicates P<0.05, Fisher LSD post hoc analysis).

FIG. 2 shows the effect of Kp-10 (SEQ ID NO: 14) and the modified Kp-10derived peptides 6 and 8 on PPI in mice treated with MK-801. C57BL/6Jmice were injected with a peptide dissolved in DMSO and diluted in PBSto a final concentration of 0.2% (w/v) DMSO, MK-801 was injected after15 min, and 15 min later measurement of the PPI was performed at 69, 73,78 and 81 decibels. Mice were treated with (from left to right for eachintensity): 0.2% DMSO in PBS (control, black bars, n=25), MK-801 alone(0.1 mg/kg, white bars, n=12), MK-801 and Kp-10 (13 μg/mouse, light graybars, n=28), MK-801 and Peptide 6 (SEQ ID NO: 6, 8.75 μg/mouse, mediumgray bars, n=16) or MK-801 and Peptide 8 (SEQ ID NO: 8, 8.75 μg/mouse,dark gray bars, n=14); Repeated measure ANOVA, F (4, 90)=8.6,P<0.0001; * indicates P<0.05, Fisher LSD post hoc analysis compared tocontrol; # indicates P<0.05, Fisher LSD post hoc analysis compared toMK-801.

FIG. 3 shows the anti-depressive effect of Peptide 6 (SEQ ID NO: 6) incomparison with Clozapine. Mice were treated with (left to right):0.003N HCl and 0.2% DMSO (control, black bars, n=8), Peptide 6 (8.75 μg,light gray bars, n=7), or Clozapine (0.3 mg/kg, dark gray bars, n=6),and subjected to a tail suspension test. Peptide 6 increased latencytime until the first occurrence of immobility (First Immobility, ANOVA,F (2, 18)=10.8, P=0.0008) and reduced the total duration of immobility(Total Immobility, ANOVA, F (2, 18)=3.95, P=0.039). * indicates P<0.05,Fisher LSD post hoc analysis compared to control; # indicates P<0.05,Fisher LSD post hoc analysis compared to Clozapine treatment.

FIGS. 4A-4G shows the therapeutic effects and lack of side effects ofPeptide 6 treatment. (A) Peptide 6 reduced the total immobility time inthe tail suspension test in a dose dependent manner. From left to right:control (0.2% DMSO), 0.0029, 0.029, 0.29, 2.9 mg/kg of Peptide 6(n=12/10/9/9/9, respectively, one way ANOVA, F (4,44)=11.3, P<0.0001).(B) PPI in mice treated with MK-801 and Risperidone, Olanzapine, orPeptide 6. From left to right: control (0.003N HCl and 0.2% DMSO, whitebars), treated with MK-801 alone (light bray bars), and treated with acombination of MK-801 and Risperidone (MK-801+Ris at 0.1 mg/kg, mediumgray bars), Olanzapine (MK-801+Ola at 0.375 mg/kg, dark gray bars) orPeptide 6 (MK-801+Pep6 at 0.29 mg/kg, black bars) (n=11/10/10/12/15,respectively, Repeated measure ANOVA, F (4,53)=2.7, P=0.03). (C) Theeffect of repeated injections of Peptide 6 on PPI at 69, 73, 78 and 81decibels. Mice were injected intraperitoneally once a day during threedays with 0.29 mg/kg of Peptide 6 and PPI was measured at the indicatedintensities 30 minutes after the third injection. White bars: control(0.2% DMSO), black bars: Peptide 6. (n=18/16. Repeated measure ANOVA, F(1,32)=4.4, P=0.04). *P<0.05, Fisher LSD post hoc analysis. (D) Theeffect of treatment with Haloperidol, Olanzapine and Peptide 6 onduration of catalepsy. From left to right: control (0.003N HCl and 0.2%DMSO, white bar), treatment with Haloperidol (HAL-1 mg/kg, light graybar), Olanzapine (OLA 0.375 mg/kg, and OLA 1.125 mg/kg, dark gray bars)and with Peptide 6 (Pep6 0.29 mg/kg, and Pep6 2.9 mg/kg, black bars),(n=7/6/7/7/7/6 respectively, *—one way ANOVA, F (5,34)=6.2, P=0.0003).(E) The effect of Haloperidol, Olanzapine and Peptide 6 on sedation.From left to right: control (0.003N HCl and 0.2% DMSO), and treated withHaloperidol (HAL-1 mg/kg), Olanzapine (OLA 0.375 mg/kg, OLA 1.125mg/kg), and Peptide 6 (Pep6 0.29 mg/kg, Pep6 2.9 mg/kg). (n=8/7/8/7/8/7respectively, one way ANOVA, F (5,39)=4.3, P<0.0001). (F) The effect ofHaloperidol, Olanzapine and Peptide 6 on spontaneous activity in theopen field. Distance—total distance in meters moved within the field.From left to right: control (0.003N HCl and 0.2% DMSO, white bar),Haloperidol (HAL-1 mg/kg, light bray bar), Olanzapine (OLA 0.375 mg/kg,OLA 1.125 mg/kg, dark gray bars), and Peptide 6 (Pep6 0.29 mg/kg, andPep6 2.9 mg/kg, black bars). (n=6/6/7/7/6/7 respectively, one way ANOVA,F (5,32)=17.8, P<0.0001). (G) The effect of Haloperidol, Olanzapine andPeptide 6 on forced activity measured as the time in seconds requiredfor each mouse to fall from a rotarod. From left to right: control(0.003N HCl and 0.2% DMSO, white bar), Haloperidol (HAL-1 mg/kg, graybar), Olanzapine (OLA 0.375 mg/kg, OLA 1.125 mg/kg, light gray bars),and Peptide 6 (Pep6 0.29 mg/kg, and Pep6 2.9 mg/kg, black bars).(n=7/6/7/7/7/6 respectively, one way ANOVA, F (5,34)=6.3, P=0.0003). *indicates P<0.05, Fisher LSD post hoc analysis compared to control; #indicates P<0.05, Fisher LSD post hoc analysis compared to MK-801.

FIG. 5. Peptide 6 administered orally reduces total immobility in atail-suspension test. 2.9 mg/kg of Peptide 6 in 100 μl water with 1%DMSO was administrated to naïve wild-type mice by oral gavage and themice were tested by a tail-suspension test (TST) 30 min later. Blackbars: control; white bars: peptide 6 (n=10/12 for control and test; *indicates P<0.02 by t-test).

FIG. 6. Effect of Peptide 6 on performance of the Alzheimer mouse model5×FAD mice in the Morris water maze. The Y axis is the time in secondswhich took the mouse to reach the platform in each day of theexperiment. Data in each day is the average of all trials in this day(n=8/8 respectively, Repeated measure ANOVA, F (1,13)=4.6, P<0.05).Black squares-control (0.1% DMSO), gray triangles—mice treated withPeptide 6. The Acquisition and the Reversal phases are described in theexperimental section. The bars in each point represent standard error.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to synthetic linear and cyclic peptideswhich are modified kisspeptin peptide Kp-10 derivatives, andpharmaceutically acceptable salts thereof.

In an attempt to develop Kisspeptin analogs with potentially improvedpharmaceutical properties, the inventors designed and prepared severalderivatives of Kp-10 (metastin amino acids 45-54), and of Kp-5 (metastinamino acids 50-54), which are described in detail below. The Kp-5sequence (the last 5 amino acids at the C-terminus of Kisspeptin) formsthe binding sequence to GPR54, while the N-terminus of the Kisspeptin isnot required for receptor binding, but may be required for stabilizationand protection from proteolytic digestion.

In certain embodiments, the synthetic peptides of the present inventionare amidated linear peptides represented by the sequenceR₁-Xaa₁-Gly-Xaa₂-Xaa₃-Xaa₄-Xaa₅-NHR₂. Xaa₁ and Xaa₂ are hydrophobicamino acids selected from phenylalanine (Phe), isoleucine (Ile), leucine(Leu), valine (Val), norleucine (Nle) and analogs thereof; Xaa₃ is abasic amino acid selected from arginine (Arg), lysine (Lys),homo-arginine (homo-Arg), homo-lysine (homo-Lys), ornithine (Orn) andanalogs of Arg; Xaa₄ is an aromatic amino acid selected from tryptophan(Trp) and analogs thereof; Xaa₅ is an aromatic amino acid selected fromtyrosine (Tyr), histidine (His), O-methyl-tyrosine and2-hydroxy-3-methyl-phenylalanine; R₁ is selected frompara-aminophenylalanine (Pap), a Pap analog of the formulaNH₂C₆H₄(CH₂)₁₋₃CO and Pap attached through the alpha-amino group thereofto a group selected from Fmoc, FMS and C₅-C₂₀ acyl, wherein said C₅-C₂₀acyl is derived from a saturated or unsaturated C₅-C₂₀ fatty acid; andR₂ is a hydrogen atom or an alkyl group having between 1 and 5 carbons(SEQ ID NO: 1).

In certain embodiments, the linear peptides of SEQ ID NO: 1 are cyclizedvia an azo bond formed between the Pap or Pap analog and the tyrosine orhistidine residues to obtain the corresponding cyclic peptides (SEQ IDNO: 2). In general, this cyclization method involves forming an azo bondconnecting the side chains of Pap residues to those of tyrosine orhistidine residues present in the linear precursors, by using an initialdiazotization step in acidic media followed by intramolecular azocyclization in a mild basic medium.

In certain embodiments, the synthetic peptides of the present inventionare amidated linear peptides represented by the sequenceR₁-Xaa₁-Gly-Xaa₂-Xaa₃-Xaa₄-NHR₂. Xaa₁ and Xaa₂ are hydrophobic aminoacids selected from phenylalanine (Phe), isoleucine (Ile), leucine(Leu), valine (Val), norleucine (Nle) and analogs thereof; Xaa₃ is abasic amino acid selected from arginine (Arg), lysine (Lys),homo-arginine (homo-Arg), homo-lysine (homo-Lys), ornithine (Orn) andanalogs of Arg; Xaa₄ is an aromatic amino acid selected from tryptophan(Trp) and analogs thereof; R₁ is 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-Fmoc (FMS); and R₂ is a hydrogen atom or an alkyl group havingbetween 1 and 5 carbons (SEQ ID NO: 3).

According to certain embodiments, the synthetic peptides are amidatedlinear peptides represented by the sequenceR₁-Xaa₁-Gly-Xaa₂-Xaa₃-Xaa₄-Xaa₅-NHR₂, the peptide is cyclic, wherein R₁is Pap, Xaa₁ is Phe, Xaa₂ is Leu, Xaa₃ is Arg, Xaa₄ is Trp, Xaa₅ is Tyror His and R₂ is H or an alkyl group having between 1 and 5 carbons (SEQID NO: 4).

In certain embodiments, the synthetic peptides of the present inventionare amidated linear peptides represented by the sequenceR₁-Xaa₁-Gly-Xaa₂-Xaa₃-Xaa₄-NHR₂, wherein R₁ is Fmoc or FMS, Xaa₁ is Phe,Xaa₂ is Leu, Xaa₃ is Arg, Xaa₄ is Trp, and R₂ is H or an alkyl grouphaving between 1 and 5 carbons (SEQ ID NO: 5).

According to certain embodiments, the synthetic peptide of the inventionis a cyclic peptide of the sequence

According to certain embodiments, the synthetic peptide of the inventionis a linear peptide of the sequence Fmoc-Phe-Gly-Leu-Arg-Trp-NH₂(Peptide 8, SEQ ID NO: 8).

In certain embodiments, the saturated fatty acids from which the C₅-C₂₀acyl is derived can be caprylic, capric, lauric, myristic, palmitic,stearic and arachidic acid, and the unsaturated fatty acids can be oleicacid, elaidic acid, linoleic acid, arachidonic acid and eicosapentaenoicacid.

According to certain embodiments, the analogs of Arg are selected fromL-2-amino-3-guanidino propionic acid and N-ω,ω-dimethyl-L-arginine; theanalogs of Trp are selected from beta-(3-benzothienyl)-L-Ala,6-methyltryptophan, 5-methoxytryptophan, 5-hydroxytryptophan,5-fluorotryptophan, 7-azatryptophpan, 5-bromotryptophan and5-methyltryptophan; the analogs of Phe are selected from2-fluorophenylalanine, 4-fluorophenylalanine, 4-bromophenylalanine,2-chlorophenylalanine, L-homophenylalanine, 4-nitrophenylalanine andα-methylphenylalanine; and the analogs of Leu or Val are selected fromt-butyl-L-alanine, L-cyclohexylglycine and L-cyclopentylglycine.

For the synthesis of the linear peptides of the invention, any methodknown in the art, such as the F-moc solid phase peptide synthesistechnique, can be used.

The synthetic peptides of the invention may be in their free form or inthe form of a salt or a chemical derivative thereof such as an ester. Asused herein, the term “salts” refers to both salts of carboxyl groupsand to acid addition salts of amino groups of the peptide molecule.Salts of a carboxyl group may be formed by means known in the art andinclude salts with inorganic salts, for example, sodium, calcium,ammonium, ferric or zinc salts, and the like, and salts with organicbases such as those formed for example, with amines, such astriethanolamine, arginine, or lysine, piperidine, procaine, and thelike. Acid addition salts include, for example, salts with mineral acidssuch as, for example, hydrochloric acid or sulfuric acid, and salts withorganic acids, such as, for example, acetic acid or oxalic acid. Theesters may be formed by reacting suitable alcohols used in peptidechemistry with terminal carboxyl groups or with free non-terminalcarboxyl groups of aspartic or glutamic acid residues.

As used herein, the phrase “Pharmaceutically acceptable” refers to anon-toxic, inert, and/or composition that is physiologically compatiblewith humans or other mammals.

According to the present invention, several modified Kp-10 derivedpeptides were prepared, in addition to the Peptides 6 and 8 of SEQ IDNOs: 6 and 8, respectively. The additional peptides have the sequencespresented below, and were found not to have a significant effect in aparadigm for assessing early information processing in naïve mice, andtherefore are used in the application for comparison only:

Sensory gating is a largely automatic process by which the brain adjustsits response to stimuli. For example, when one stimulus is presented,there is a response; however, when the first stimulus is followed by asecond stimulus soon after, the response to the second stimulus isblunted. This is an adaptive mechanism to prevent over stimulation thathelps the brain focus on a stimulus among a host of other distracters,and contributes to the ability to selectively allocate attention to asignificant event by silencing the background. The specific features ofan individual's gating processes are viewed to be plastic, and governedby genetic and developmental processes, but also by environmentalchanges, neurochemical and hormonal state of the CNS. Sensory gating wasshown to be disturbed in various disorders, including schizophrenia andAlzheimer.

Prepulse inhibition (PPI), the phenomenon by which a low-intensityprepulse stimulus attenuates the response to a subsequentstartle-eliciting stimulus, is used as a measurement of the sensorygating function (Swerdlow and Geyer, 1998).

It has been found, in accordance with the present invention, thatinjecting mice treated with MK-801, which induces psychotic symptomsmimicking the effect of schizophrenia, with Peptide 6 (SEQ ID NO: 6) orPeptide 8 (SEQ ID NO: 8) of the invention, or with Kp-10 (SEQ ID NO:14), which has previously been found to improve PPI in mouse models forschizophrenia, significantly improved PPI compared to animals treatedonly with MK-801. Furthermore, treatment with Peptide 6 restored PPIback to normal levels, having a greater effect than that of Kp-10, or ofPeptide 8 (FIG. 2).

It has additionally been found, in accordance with the presentinvention, that the effect of Peptide 6 on PPI in the MK-801schizophrenia mouse model is greater than that of known antipsychoticdrugs—Risperidone and Olanzapine (FIG. 4B). Additionally, Peptide 6 didnot have side effects typical to the anti-psychotic drugs such asHaloperidol and Olanzapine (FIGS. 4D-4G).

The present invention thus provides a synthetic peptide of the inventionas defined above or a pharmaceutically acceptable salt thereof, for useas a medicament.

The present invention further provides a pharmaceutical compositioncomprising a peptide of the invention as defined above or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.

PPI deficits are not unique to a single form of psychopathology. Inaddition to schizophrenia, impairment of PPI has been reported inseveral diseases such as autistic disorder, Huntington's chorea,obsessive-compulsive disorder, attention deficit hyperactivity disorder(ADHD), Tourette's syndrome, and some cases of Alzheimer's disease(Castellanos et al. 1996; Ornitz et al. 1992; Perry et al., 2007;Swerdlow et al. 1993; Swerdlow et al. 1995; Ueki et al., 2006).Therefore, PPI is often used as a paradigm, for assessing the modulationof early information processing and is widely used to investigateantipsychotic drug action in animals.

Thus, according to certain embodiments, the synthetic peptides of theinvention are useful for treating a disease or disorder presentingbehavioral abnormalities associated with impairment of sensory gatingfunction. According to certain embodiments, such diseases areschizophrenia, autistic disorder, Huntington's chorea,obsessive-compulsive disorder, attention deficit hyperactivity disorder(ADHD) or Tourette's syndrome. According to a certain embodiment, thedisease is schizophrenia. In certain embodiments, the synthetic peptidehas a sequence as set forth in SEQ ID NO: 6. In certain embodiments, thesynthetic peptide has a sequence as set forth in SEQ ID NO: 8.

The phrase “disease or disorder presenting behavioral abnormalitiesassociated with impairment of sensory gating function” is used herein torelate to any disease or disorder in which any association with impairedsensory gating as described above, such as for example, impaired PPI,has been found.

It has further been found, in accordance with the present invention,that Peptide 6 improves spatial learning and memory in the Morris WaterMaze in a mouse model for Alzheimer's disease (FIG. 6).

According to certain embodiments, the synthetic peptides of theinvention may be used for treating Alzheimer's disease. According to acertain embodiment, the synthetic peptide is Peptide 6.

It has further been shown, in accordance with the present invention,that Peptide 6 has an anti-depressive effect in the tail suspension testin Naïve animals, (FIGS. 3 and 4A), and that effect was higher than thatof the known anti-psychotic drug clozapine. Peptide 6 additionally hasbeen found to have an effect of increasing PPI in Naïve animals (FIGS.1C and 4C).

Thus, according to certain embodiments, the synthetic peptides of theinvention may be used for treating depression and/or cognitiveimpairment. According to a certain embodiment, the synthetic peptide isPeptide 6.

Depression as well as cognitive impairment are among the known symptomsof schizophrenia and of Alzheimer's disease.

Thus, according to certain embodiments, the synthetic peptides of theinvention may be used for treating depression and/or cognitiveimpairment associated with schizophrenia or with Alzheimer's disease.

In certain embodiments, the synthetic peptides of the invention may beused for treating depression and/or cognitive impairment associated withdiseases other than schizophrenia and Alzheimer's disease.

In certain embodiments, the peptide is Peptide 6, having a sequence asset forth in SEQ ID NO: 6.

According to certain embodiments, the pharmaceutical compositions of theinvention may comprise a single synthetic peptide of the invention ormore than one synthetic peptides of the invention.

The pharmaceutical compositions of the present invention may be preparedby conventional techniques, e.g. as described in Remington: The Scienceand Practice of Pharmacy, 19^(th) Ed., 1995. The composition may be insolid, semisolid or liquid form or it can be designed for slow releaseof the peptide. According to certain embodiments, the compositions ofthe invention may be further mixed with excipients such as stabilizers,flavoring agents, antiseptics, binders, antioxidants, etc.

As used herein, a “pharmaceutically acceptable carrier” is apharmaceutically acceptable solvent, suspending agent or vehicle, fordelivering the instant compounds to the patient. The carrier may beliquid or solid and is selected with the planned manner ofadministration in mind. Liposomes can also be a pharmaceutical carrier.

The present invention further provides a method for the treatment of adisease or disorder presenting behavioral abnormalities associated withimpairment of sensory gating function, depression or cognitiveimpairment, comprising administering to a patient in need thereof aneffective amount of a synthetic peptide of the invention or apharmaceutically acceptable salt thereof.

According to certain embodiments, the disease is schizophrenia.According to certain embodiments, the disease is Alzheimer's disease.According to certain embodiments, the synthetic peptide is Peptide 6.

As used herein, the phrase “effective amount” or “therapeuticallyeffective amount” refers to the quantity of a component that issufficient to yield a desired therapeutic response without undue adverseside effects (such as toxicity, irritation, or allergic response)commensurate with a reasonable benefit/risk ratio when used in themanner of this invention.

As used herein, the term “treatment” includes ameliorating, alleviating,attenuating and/or even abolishing symptoms associated with the diseaseor disorder. In case of schizophrenia, it is contemplated by theinvention that the treatment with the synthetic peptides of theinvention will ameliorate/alleviate the psychotic and/or depressionsymptoms associated with the disease and/or will improve the cognitivefunctions impaired in the schizophrenic patient.

According to certain embodiments, the treatment of schizophreniacomprises administration of a peptide of the invention or apharmaceutically acceptable salt thereof alone or in combination withone or more drugs suitable for treatment of schizophrenia such asclozapine, amisulpride, olanzapine, risperidone, quetiapine,ziprasidone, aripiprazole or paliperidone.

According to certain embodiments, the treatment of Alzheimer's diseasecomprises administration of a peptide of the invention or apharmaceutically acceptable salt thereof alone or in combination withone or more drugs suitable for treatment of Alzheimer's disease such asacetylcholinesterase inhibitors (e.g. tacrine, rivastigmine, galantamineand donepezil) or NMDA receptor antagonists such as memantine, or firstgeneration antipsychotics such as Chlorpromazine, Haloperidol,Perphenazine, Fluphenazine.

Any suitable composition and route of administration is encompassed bythe invention, including oral (e.g. in the form of tablets, capsules,microcapsules, and the like), parenteral (e.g., in the form ofsubcutaneous, intramuscular, intraarticular, or intravenous injection),inhalation, intranasal (in the form of sprays), intrathecal,intraperitoneal, intradermal, transdermal or other known routes ofadministration suitable for the administration of peptides to humanbeings.

According to certain embodiments, the peptides of the invention areadministered by oral administration.

The dose of the peptide to be administered will be determined by thecompetent physician and will depend on the agent used, the severity ofthe disease, the age, and the weight of the patient, and may vary from0.1-100 mg, preferably 1.0-50 mg, more preferably 1.0-20 mg for oralapplication; or from 0.01-30 mg, preferably 1.0-30 mg, more preferably1.0-20 mg for parenteral application; or from 0.01-10 mg, preferably0.05-5 mg, more preferably 0.05-0.2 mg for intravenous (IV) application.

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES

Materials and Methods

Unless otherwise indicated, Peptide 6 (cKp-7,Pap-Phe-Gly-Leu-Arg-Trp-Tyr-NH₂ Pap=p-aminophenylalanine; the Pap andthe Tyr are connected in an internal azo-bridge, to yield a cyclicpeptide (GL Biochem (Shanghai) Ltd.)) was dissolved in DMSO and dilutedin PBS to a final concentration of 0.1% DMSO, and 100 μl were injectedintraperitoneally at the indicated dose 30 min before the behavioralanalysis.

Risperidone (Sigma, Israel) was dissolved in 0.06N HCL and administeredintraperitoneally at a dose of 0.1 mg/kg in a final concentration of0.003N HCL, 0.1% DMSO, 30 min prior to the PPI analysis.

Olanzapine (Sigma, Israel) was dissolved in 0.06N HCL and administeredintraperitoneally at a dose of 0.375 or 1.125 mg/kg in a finalconcentration of 0.003N HCL, 0.1% DMSO, 30 min prior to the behavioranalysis.

Haloperidol (Sigma, Israel) was dissolved in 0.06N HCL and administeredintraperitoneally at a dose of 1 mg/kg in a final concentration of0.003N HCL, 0.1% DMSO, 30 min prior to the behavior analysis.

Animals.

Inbred 12-16 week old C57Bl/6 mice were supplied by the Animal BreedingCenter of the Weizmann Institute of Science. Since the estrous cycleaffects PPI, male mice were used in all experiments unless otherwiseindicated. All animals were handled according to the regulationsformulated by the Weizmann Institute's Animal Care and Use Committee andmaintained in a pathogen free environment.

Kp-10 Peptide.

Kisspeptin-10 (Kp-10, human Metastin amino acids 45-54, YNWNSFGLRF-NH₂(SEQ ID NO: 14), Weizmann Peptide Facility or Sigma-Aldrich, Israel) wasinjected intraperitoneally at a dose of 13 μg/mouse dissolved in PBS, 30min before the PPI analysis. MK-801 (Sigma) was used at 0.1 mg/kg andadministered 15 min prior to the PPI analysis.

Acoustic Startle Response (Prepulse Inhibition, PPI) Testing.

Acoustic startle response testing was performed within startle chamberspurchased from Med Associates, Inc. (Med Associates, St. Albans, Vt.,USA), as described in Cardon et al., 2010. During a period ofacclimation, a 65-decibel (dB) background noise was presented for 5-min,and continued throughout the test session. All sessions for testing ofacoustic startle response consisted of startle trials (pulse alone,40-milliseconds (ms), 120-dB), prepulse trials (prepulse 20-ms, 69, 73,78, or 81 dB followed by a [100-ms delay] pulse), and no-stimulustrials. All sessions were presented in pseudo-random order. The averagetime between trials was 15 seconds (range 12-30 seconds). Startleresponse was calculated from the reaction to the first pulse-alonetrial.

Calculation of Prepulse Inhibition (PPI).

PPI was calculated as: % PPI=100−{[(startle response forprepulse+pulse)/(startle response for pulse-alone)]×100}. Reduced PPIindicates schizophrenia-related behavior. No difference between Kp-10and PBS treated mice was observed in the startle response and in theprepulse-alone trials, in which the response was negligible compared tothe pulse trials.

Tail Suspension Test.

The tail suspension apparatus was made of horizontal metal rod supportedby a stand. Each mouse was suspended from its tail using adhesive tape,suspending each mouse individually. The immobility time was measuredduring a 6 min observation period, latency to first immobility and totalimmobility is calculated.

Open-Field Test.

The open-field apparatus consisted of a white Plexiglas box (40×40×30cm) with 16 squares (10×10 cm) painted on the floor (12 outer and 4inner). Each mouse was placed in the center of the apparatus to initiatea 15-min test session. The number of entrances to the inner squares,time (min) spent in the inner squares, and total distance moved withinthe field (cm) were monitored.

Radial Arm Water Maze (RAWM).

Mice were tested for two days on 6 radial arm water maze paradigm in awater pool, as described in Alamed et al. (2006). Briefly, mice receivedinjection of a peptide of the invention in the beginning of each day. Onthe first day, 15 trials were performed, in trials 1, 3, 5, 7, 9 and 11the platform was visible, and in all other trials the platform washidden. On the second day, 12 trials were performed, all of them withhidden platform. The time required to reach the platform and the numberof errors (incorrect arm entries) in 1-min time period was recorded.

Morris Water Maze (MWM) Test.

Acquisition phase: mice were given 3-4 trials per day on 6 consecutivedays. In each trial they were required to find a hidden platform located1.5 cm below the water surface in a pool 1.4 m in diameter. Within thetesting room, only distal visuo-spatial cues for location of thesubmerged platform were available. The escape latency, i.e., the timerequired by the mouse to find the platform and climb onto it, isrecorded for up to 60 s. Each mouse is allowed to remain on the platformfor 20 s and is then moved from the maze to its home cage. If the mousedoes not find the platform within 60 s, it is placed manually on theplatform and returned to its home cage after 20 s. The interval betweentrials was 600 s. On day 6 each mouse was tested by a probe trial: theplatform was removed from the pool and the location of each mouse in thepool was followed for 60 sec. For the reversal phase, mice were givenfour trials per day on 2 consecutive days, and the platform was at adifferent location. Data were recorded using an EthoVision automatedtracking system (Noldus).

Catalepsy and Sedative Effect.

Catalepsy is a state of immobility, and was measured by means of the bartest. The forepaws of the animal were placed on a bar (0.75 cm indiameter and 5 cm above the floor) and the latency until the mouse movedthe forepaws off the bar was measured.

Sedative effect was evaluated by observing the mice and scoring theirsedative state as follows: 0, spontaneous movement; 1, mild intermittentspontaneous movement; 2, severe intermittent spontaneous movement; 3, nospontaneous movement; 4, loss of auditory reflex; 5, loss of cornealreflex; 6, loss of response to tail pinch.

Forced activity (Rotarod) was analyzed using the Rotarod apparatus(Jones and Roberts 7650, Ugo Basile, Italy). Animals were placed on anaccelerating rod, and time required for each mouse to fall from the rodwas recorded. Out of three trails, the longest time taken for an animalto fall from the rod was recorded and used for analysis.

Example 1: Synthesis of Modified Kp-10 Derived Peptides

Linear Peptides 8 (SEQ ID NO: 8), and linear Peptide 4 (SEQ ID NO: 7)which is a precursor of cyclic Peptide 6 having the same sequence (SEQID NO: 6), were synthesized by the solid-phase method using an AdvancedChemical APEX 396 multiple peptide synthesizer (Louisville, Ky.)following the commercial protocol recommended by the manufacturer forthe Fmoc-strategy. After completion of peptide chain assembly, Peptide 4was deprotected and cleaved from the polymeric support (Rink amide) witha mixture of triflouroacetic acid (TFA), thioanisol triethylsilane andwater (85:5:5:5 v/v) at room temperature for 3 hours. Peptide 8 wascleaved from the polymeric carriers while leaving the N-terminalFmoc-protection intact. The crude peptides, following precipitation witht-butylether, were purified by HPLC (to >95%).

Peptide 4 was cyclized to form an internal azo-bond between the Pap andTyr residues, following reaction with an equimolar amount of nitrousacid (HNO₂) at acidic pH ˜1 at 0° C., and cyclization at high dilution(1 mg/2 ml) upon adjustment of the pH to 8, thereby obtaining Peptide 6.The cyclization was nearly quantitative. Final purification (>95%) byHPLC led to the desired cyclic product as revealed by mass spectrometryand amino acid analysis. The cyclization procedure was according toFridkin et al., 2006 and Fridkin et al., 2011.

Linear Peptides 1 and 3 (SEQ ID NOs: 9 and 11, respectively), having thesame sequence as cyclic Peptides 2 and 5, respectively, were synthesizedby the solid-phase method as described above, and titrated withpotassium ferrocyanide, in a solution of ammonium acetate pH 7 to formthe corresponding cystine (S—S) cyclic Peptides 2 and 5 (SEQ ID NOs: 10and 12, respectively).

Peptide 7 (SEQ ID NO: 13) was synthesized as described above for Peptide8.

Example 2: Modified Kp-10 Derived Peptides Improve PPI in Naïve Mice

To determine the effect of the Kp-10 derivatives on behavior, we studiedthe effect of each of the peptides 2, 5, 6, 7 and 8 on PPI.

To test Peptide 2 (SEQ ID NO: 10), the peptide was dissolved in DMSO andthen diluted in PBS to a final concentration of 1% (w/v) DMSO. MaleC57Bl/6J naïve mice were injected intraperitoneally with 15 μg of thepeptide or with 1% DMSO in PBS (control). Prepulse intensity analysiswas performed after 30 minutes at 69, 73, 78 or 81 decibels. As can beseen from FIG. 1A, Peptide 2 slightly improved the % PPI relative tocontrols, however, the effect was not significant (Repeated measureANOVA, F (degrees of freedom) (1,18)=4.2, P=0.06).

To test Peptide 5 (SEQ ID NO: 12), mice were similarly injected with 8.3μg Peptide 5 in 1% DMSO in PBS or with 1% DMSO in PBS (control).Prepulse intensity analysis was performed after 30 minutes at 69, 73, 78or 81 decibels. As can be seen from FIG. 1B, Peptide 5 slightly improvedthe % PPI at 73, 78 and 81 decibels relative to controls, however, theeffect was not significant (Repeated measure ANOVA, F (1, 12)=0.1,P=0.7).

To test Peptide 6 (SEQ ID NO: 6), mice were injected with 8.75 μgPeptide 6 in 1% DMSO in PBS or with 1% DMSO in PBS (control). Prepulseintensity analysis was performed after 30 minutes at 69, 73, 78 or 81decibels. As can be seen from FIG. 1C, Peptide 6 strongly elevated the %PPI at all intensities relative to controls, and this effect wassignificant at 78 decibels (Repeated measure ANOVA, F (1, 19)=4.7,P=0.047*P<0.05, Fisher LSD post hoc analysis).

To test Peptide 7 (SEQ ID NO: 13), mice were injected with 15.3 μgPeptide 7 in 1% DMSO in PBS or with 1% DMSO in PBS (control). Prepulseintensity analysis was performed after 30 minutes at 69, 73, 78 or 81decibels. As can be seen from FIG. 1D, Peptide 7 slightly improved the %PPI at 73, 78 and 81 decibels relative to controls, however, the effectwas not significant (Repeated measure ANOVA, F (1, 18)=1.15, P=0.3).

To test Peptide 8 (SEQ ID NO: 8), mice were injected with 8.75 μgPeptide 8 in 1% DMSO in PBS or with 1% DMSO in PBS (control). Prepulseintensity analysis was performed after 30 minutes at 69, 73, 78 or 81decibels. As can be seen from FIG. 1E, Peptide 8 strongly elevated the %PPI at all intensities relative to controls, and this effect wassignificant at 73 decibels (Repeated measure ANOVA, F (1, 31)=5.3,P=0.028*P<0.05, Fisher LSD post hoc analysis).

To summarize, as can be seen from FIGS. 1A-1E, while treatment withPeptides 2, 5 and 7, did not significantly improve the PPI responserelative to untreated animals, administration of Peptides 6 and 8resulted in a significant increase in % PPI at all intensities.

Example 3: Modified Kp-10 Derived Peptides Protect Against PPIDysfunction in an Animal Model for Schizophrenia

To determine if the peptides that most strongly affect PPI in naïve mice(Peptides 6 and 8) can abolish psychotic symptoms in a drug inducedanimal model of schizophrenia, we analyzed their effect on PPI in theMK-801 model. MK-801 (an antagonist of the N-methyl-D-aspartate (NMDA)receptor channel) acts as a psychomimetic agent, inducing psychoticsymptoms that mimic the cognitive impairment and behavioralabnormalities associated with schizophrenia.

The neurotransmitter imbalance induced by MK-801 causes PPI dysfunction,one of the characteristic features of patients with schizophrenia. In anexperiment depicted in FIG. 2, C57BL/6J mice were injected with Kp-10(13 μg/mouse, light gray bars), or with Peptides 6 (8.75 μg/mouse,medium gray bars) or 8 (8.75 μg/mouse, dark gray bars) dissolved in DMSOand then diluted in PBS to a final concentration of 0.2% (w/v) DMSO; thecontrol group received 0.2% DMSO in PBS (black bars). After 15 min,MK-801 (0.1 mg/kg) was injected; 15 min later, measurement of the PPIwas performed. As expected, in mice injected with MK-801 (white bars),the PPI response was reduced compared to the PBS injected control. Allthree treatments (Kp-10, Peptide 6 and Peptide 8) significantly improvedPPI compared to the MK-801 treated animals (FIG. 2, repeated measureANOVA, F (4, 90)=8.6, P>0.0001; # indicates P<0.05, Fisher LSD post hocanalysis compared to MK-801). However, only Peptide 6 restored PPI backto normal levels (* in FIG. 2 indicates P<0.05, Fisher LSD post hocanalysis compared to control, no significant difference at all prepulseintensities found between no MK-801 controls and treatment with Peptide6); Kp-10 and Peptide 8 improved PPI after MK-801 administration but didnot abolish its affect, and PPI in these groups remained significantlydifferent from that of the control group. Thus, peptide 6 is moreeffective in abolishing the effect of MK-801 on PPI than either Peptide8 or Kp-10.

Example 4. Antidepressive Effect of Modified Kp-10-Derived Peptides

Schizophrenia patients suffer from cognitive and negative symptoms(e.g., depression) in addition to psychotic symptoms. Kp-10 was shown toelevate BDNF mRNA levels in hippocampal slices (Arai et al., 2009), andwas further shown in WO 2010/137022 to have an antidepressant effect bythe tail suspension test, an established method for screeningantidepressant-like activity in mice (Steru et al., 1985; Cryan et al.,2005).

Peptide 6 of the invention was tested for an antidepressive effect bythe tail suspension test as follows: Naïve mice were treated withPeptide 6 of the invention (8.75 μg of peptide dissolved in DMSO andthen diluted in PBS to a final concentration of 0.2% DMSO (w/v), HCL wasadded to a final concentration of 0.003N) or with Clozapine (0.3 mg/kgdissolved in 0.3N HCL diluted in PBS to a final concentration of 0.003NHCL, DMSO was added to a final concentration of 0.2% DMSO (w/v)) andsubjected to the tail suspension test, in which the mouse was suspendedby the tail from a lever, and the movements of the animal were recorded.As can be seen in FIG. 3, treatment with Peptide 6 (light gray bars)significantly increased latency until the first occurrence of immobilitycompared with the control (black bars) and with Clozapine (dark graybars) (ANOVA, F (2, 18)=10.8, P=0.0008) and significantly reduced thetotal duration of immobility (ANOVA, F (2, 18)=3.95, P=0.039) comparedwith the control and with Clozapine treatment. These results indicatethat Peptide 6 of the invention can be used as an antidepressant andthat it has a higher antidepressive activity than the antipsychotic drugClozapine.

Example 5. Therapeutic Effects and Lack of Side Effects of Peptide 6

To determine the dose effect of Peptide 6 we injected it in increasingdosages and analyzed its effect on tail suspension test. Peptide 6reduced the total immobility time in a dosage dependent manner, at0.029, 0.29 and 2.9 mg/kg (FIG. 4A). This result showed that Peptide 6has a wide therapeutic range. A dose of 0.29 mg/kg is equivalent to thedose used for Kp-10, which reduced the total immobility in the tailsuspension test (FIG. 10C of WO 2010/137022).

We further analyzed the effect of Peptide 6 on the reduction in PPIinduced by MK-801 in comparison with the common antipsychoticsRisperidone and Olanzapine. Both of these drugs cause sedative effects,and since PPI cannot be measured correctly in sedated mice we firstevaluated the highest dose of the drugs that can be injected to the micewithout causing sedative effects: the chosen dose was 0.1 mg/kg forRisperidone and 0.375 mg/kg for Olanzapine (data not shown). As can beseen from FIG. 4B, all drugs elevated PPI in the MK-801 treated mice,but only Peptide 6 (black bars) completely overcame the effect of MK-801and restored the PPI levels to control level (white bars). ANOVA ofpercent PPI indicated a significant main effect of the treatments, butnot of the prepulse intensities. One-way ANOVA analysis of singleintensities revealed statistically significant differences betweentreatments at prepulse intensities of 69, 73 and 81 dB.

The effect of repeated injections of Peptide 6, was tested by injecting0.29 mg/kg of Peptide 6 once a day during three days and measuring PPI30 minutes after the third injection. As can be seen from FIG. 4C,repeated injections of Peptide 6 increased the PPI at all intensities,the effect was significant at PPI of 69 and 73 decibels.

To evaluate the potential side effect of Peptide 6 we analyzed itseffect on catalepsy, sedative effect, and effect on spontaneous (openfield) and forced (rotarod) activity as compared with the antipsychoticsHaloperidol (1 mg/kg) and Olanzapine (0.375 and 1.125 mg/kg).

Halperidol (light gray bar, HAL) and Olanzapine (dark gray bars, OLA at0.375 and 1.125 mg/kg) both had catalepsy effects even at low levels ofOlanzapine, on the other hand, Peptide 6 (black bars, Pep6 at 0.29 and2.9 mg/kg) did not have catalepsy effect at either dosage tested (FIG.4D).

Next we analyzed the sedative effects of Haloperidol, Olanzapine andPeptide 6. Sedative effect was evaluated by observing the animal using asedative score. Haloperidol had low sedative effect, Olanzapine hadsedative effect at a dose of 1.125 mg/kg, Peptide 6 did not have asedative effect at either of the two dosages tested (FIG. 4E).

Regarding spontaneous and forced activity, Haloperidol (HAL, 1 mg/kg,light gray bar) and Olanzapine (OLA 0.375 and OLA 1.125 mg/kg, dark graybars) reduced mice activity both in the open field (spontaneous) and inthe Rotarod (forced), while Peptide 6 (Pep6 at 0.29 and 2.9 mg/kg, blackbars) did not affect activity level in either of the tests at eitherdosage tested (FIGS. 4F,G). These results showed that Peptide 6 has nomotor side effect compared to other antipsychotic drugs.

Example 6. Peptide 6 Administered Orally Reduces Total Immobility in aTail-Suspension Test

2.9 mg/kg of Peptide 6 were dissolved in DMSO and then diluted in waterto a final concentration of 1% DMSO. 100 μl was administrated to naïvewild-type mice by oral gavage and the mice were tested by atail-suspension test (TST) 30 min later. Control mice received 100 μlwater with 1% DMSO. As can be seen from FIG. 5, Peptide 6 significantlyreduced the total immobility relative to the control.

Example 7. Plasma Stability of Peptide 6

1 μM Peptide 6 was dissolved in human plasma, and peptide concentrationwas analyzed by HPLC after 0, 0.5, 1, 1.5, 2 h of incubation. The assaywas performed by Cerep, Inc (Redmond, Wash., U.S.A). As presented inTable 1, Peptide 6 is highly stable, with a calculated half-life of >120minutes. Peptide 6 exhibits increased stability, with 94% of theoriginal material remained in the human plasma after 60 min, relative toKp-10, which was below detectable level after 60 min (chan, et al.,2011, Kisspeptin resets the hypothalamic GnRH clock in men, J Clin.Endocrinol. Metab. 96(6):E908-15).

TABLE 1 Plasma stability of Peptide 6 Incubation Time (minutes) % MeanCompound Remaining 0 100.0 30 118.9 60 93.7 90 97.2 120 71.8

Example 8. Effect of Peptide 6 on Spatial Learning and Memory in anAlzheimer Mouse Model 5×FAD Transgenic Mice

5×FAD mice overexpress the [APP K670N/M671L (Swedish)+I716V(Florida)+V717I (London) and PSI M146L+L286V]. The generation of the5×FAD mice has been described previously (Oakley, et al., 2006, JNeurosci 26(40):10129-40, 2006).

The effect of Peptide 6 on spatial learning and memory was tested in theMorris water maze behavioral test. At the acquisition phase, mice weregiven three to four trials per day on 6 consecutive days. In each trial,the mice were required to find a hidden platform located 1.5 cm belowthe water surface in a 1.1-m-diameter pool.

Within the testing room, only distal visual-spatial cues for location ofthe submerged platform were available. The escape latency, i.e., thetime required by the mouse to find the platform and climb onto it, wasrecorded for up to 60 sec. Each mouse was allowed to remain on theplatform for 20 sec and was then moved from the maze. If the mouse didnot find the platform within 60 sec, it was manually placed on theplatform for 20 sec. The interval between trials was 10 min. In theprobe trial phase, the platform was removed from the pool and thelocation of each mouse in the pool was followed for 60 sec. Data wererecorded using an EthoVision automated tracking system (Noldus).

For the reversal phase, mice were given four trials per day on 2consecutive days, in this phase the platform was at a differentlocation. Mice were either not treated (control), or treated with 8.75μg/mouse of Peptide 6 by an intraperitoneal injection 30 minutes beforetesting. As seen from FIG. 6, mice treated with Peptide 6 (graytriangles) took less time than untreated mice (black squares) to findthe platform on days 5 and 6 of the acquisition phase and days 1 and 2of the reversal phase.

Overall the above result demonstrate that Peptide 6 is a potential drugcandidate with higher efficiency and increased stability compared withKp-10, wide therapeutic window and high tolerability.

REFERENCES

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What is claimed is:
 1. A method for the treatment of a disease ordisorder selected from the group consisting of schizophrenia, autisticdisorder, Huntington's chorea, obsessive-compulsive disorder, attentiondeficit hyperactivity disorder (ADHD), Alzheimer's disease (AD),Tourette's syndrome, depression and cognitive impairment, the methodcomprising administering to a patient in need thereof an effectiveamount of a synthetic linear peptide of the sequence: (SEQ ID NO: 8)Fmoc-Phe-Gly-Leu-Arg-Trp-NH₂:

or a pharmaceutically acceptable salt thereof, thereby treating thedisease or disorder.
 2. The method of claim 1, wherein said disease ordisorder is Alzheimer's disease (AD).
 3. The method of claim 1, whereinsaid depression and/or said cognitive impairment are associated withschizophrenia or with Alzheimer's disease.
 4. The method of claim 1,wherein said administering is by oral administration.
 5. The method ofclaim 1, wherein said patient is a human being.
 6. A method for thetreatment schizophrenia, the method comprising administering to apatient in need thereof an effective amount of a synthetic linearpeptide of the sequence: Fmoc-Phe-Gly-Leu-Arg-Trp-NH₂ (SEQ ID NO: 8) ora pharmaceutically acceptable salt thereof, thereby treating theschizophrenia.